Orthogonal analysis of dystrophin protein and mRNA as a surrogate outcome for drug development

Abstract

Aim: Detection of drug-induced dystrophin in patient muscle biopsy is a surrogate outcome measure for Duchenne muscular dystrophy. We sought to establish and validate an orthogonal approach to measurement of dystrophin protein and RNA in muscle biopsies. Materials & methods: Validated methods were developed for dystrophin western blotting, mass spectrometry, immunostaining and reverse transcriptase PCR of biopsy mRNA using muscle biopsy standards. Results: Both western blotting and mass spectrometry validated methods demonstrated good linearity, and acceptable precision and accuracy with a lower limit of quantitation at 1%. Immunostaining and reverse transcriptase PCR methods were shown to be reliable. Conclusion: The described orthogonal approach is sufficient to support measures of dystrophin as a surrogate outcome in a clinical trial.

Keywords: Duchenne muscular dystrophy; dystrophin; exon-skipping; muscle; oligonucleotide drug; phosphorodiamidate morpholino oligomer; surrogate biomarker.